Blood donor screening with cobas s 201/cobas TaqScreen MPX under routine conditions at German Red Cross institutes.

BACKGROUND: In 1997 the German Red Cross (GRC) blood donor services introduced mini-pool nucleic acid testing (NAT) for human immunodeficiency virus (HIV)-1, hepatitis C virus (HCV) and hepatitis B virus (HBV) to increase blood safety. With the new cobas s 201/cobas TaqScreen MPX, a fully automated extraction method and a multiplex amplification system specifically adapted to the needs of blood donation services is available. METHODS: The cobas s 201 system was evaluated at the GRC BTS locations Hagen, Springe and Frankfurt. In phase A, the analytical sensitivity for the detection of HBV, HCV and HIV-1 was investigated and in phase B, at least 60,000 samples at each test site were screened in parallel with the MPX test on s 201 system and the existing routine mini-pool NAT system to compare the diagnostic specificity and the diagnostic sensitivity. RESULTS: Comparable analytical sensitivities in a range of 1.6-3.6 IU/ml, 4.9-10.9 IU/ml and 14.7-26.6 IU/ml for HBV, HCV HIV, respectively, for the MPX test on s 201 system (95% probability based on probit analysis) were determined at all test sites. The diagnostic sensitivity was 99.8% and the diagnostic specificity was 99.85%. CONCLUSIONS: The MPX test on s 201 system is a fully automated NAT system suitable for routine blood donor screening. The analytical sensitivity as well as the diagnostic sensitivity fulfilled all requirements of the Paul Ehrlich Institute for blood donor screening in mini-pools up to 96 donations per pool. A major benefit of the automated NAT system is the reduced personnel time and the extensive complete barcode-controlled process documentation.

PCR for HBV, HCV and HIV-1 experiences and first results from a routine screening programme in a large blood transfusion service.

We adapted the PCR method to screen up to 3000 blood donations per day for hepatitis B, hepatitis C and HIV-1 virus contamination. Up to 600 aliquots (average 418 donations) are pooled by using an automatic sample processor with disposable tips (validated to avoid contamination) taken from blood donations which are serologically negative and free for clinical usage according to federal regulations. In the case of a positive PCR pool result the viraemic donation is identified by two additional PCR pools testing steps with smaller pool sizes. All of the steps are supported by electronic data processing. After virus concentration by ultracentrifugation, and in the case of HCV and HIV-1 an additional reverse transcription step, PCR amplifications are performed. PCRs are done for each virus in two genomic regions. Laser-induced detection after PAGE and computer-analysis are used to identify the amplification products. Using this validated methodology routine we have checked 428 896 donations up to the end of August 1996. During this survey we found at least 24 viruses-containing donations which were negative in corresponding serological tests and would have been transfused (2 HBV-, 22 HCV-, 0 HIV-1 -containing donations). It seems possible for large transfusion centres to shorten the diagnostic window periods with our PCR-methodology with acceptable costs (15 DM per donation for all three viruses including logistics, developments and investments).

[Routine PCR screening for HBV, HCV and HIV-I genome in a large blood donation services--experiences and initial results]. / PCR-Routine-Screening auf HBV-, HCV- und HIV-I-Genom in einem grossen Blutspendedienst--Erfahrungen und erste Ergebnisse.

We adapted the PCR method to screen up to 3,000 blood donations per day for HBV, HCV, and HIV-1 contamination. Concerning logistics: The first step is the generation of 3 identical microtiter plates (PT) by using the self-validated automatic sample processor with disposable tips. Using the first PT, we pooled up to 600 aliquots taken from blood donations which are serological negative and free for clinical usage according to actual federal regulations. In the case of a positive PCR pool result the viremic donation is identified by 2 additional PCR pool testing steps with smaller pool sizes using the second and third PT. All described steps are supported by electronic data processing. The PCR-method: After virus concentration by ultracentrifugation and--in case of HCV and HIV-1--additional reverse transcription PCR-amplifications were performed. PCR in two genomic regions are done for each virus. Laser-induced fluorescence detection after polyacrylamide gel electrophoresis and computer analysis were used to check the amplification products. Using this approach, a virus-containing donation can be detected in up to 599 negative samples with following sensitivity: HBV and HIV-1, 1,000-1,500 genome equivalents/ml; HCV, 2,000-2,500 genome equivalents/ml, thus sensitivity being in the range of commercial available PCR kits when testing nonpooled samples. The sensitivity was validated by using national and international available standards with known virus genome concentrations. All processing steps are checked using different controls such as, e.g., negative, positive, premix controls, reporter virus, inhibition controls. Routinely employing this validated methodology, we investigated 327,013 donations until the end of June 1996. During this survey, we found at least 16 virus-containing donations which are negative in corresponding serological tests and would have been transfused (4 HBV-, 13 HCV-, 0 HIV-1-containing donations), including one later seroconversion for HBV and one for HCV. Using our adapted PCR-methodology, it seems possible to shorten the diagnostic window periods with acceptable costs for large transfusion centers (15 DM per donation for all 3 viruses including all steps and investments). Therefore, PCR seems to become a new method of choice to prevent transfusion transmitted infections.